1-Propanaminium, N,N,N-trimethyl-3-(octadecyloxy)-, chloride (1:1)

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 July 2007 to 29 August 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 30 August 2005 Date of Signature: 21 November 2005

Test material

Test material information

Constituent 1

Reference substance name:

trimethyl[3-(octadecyloxy)propyl]azanium chloride

EC Number:

700-414-8

Cas Number:

23328-71-4

Molecular formula:

C24H52ClNO

IUPAC Name:

trimethyl[3-(octadecyloxy)propyl]azanium chloride

Details on test material:

- Name of test material (as cited in study report): 3-Octadecyloxypropyl-N,N,N-trimethylammonium chloride
- Molecular formula (if other than submission substance): Not applicable
- Molecular weight (if other than submission substance): Not applicable
- Smiles notation (if other than submission substance): Not applicable
- InChl (if other than submission substance): Not applicable
- Structural formula attached as image file (if other than submission substance): Not applicable
- Substance type: white solid
- Physical state: solid
- Analytical purity: Not reported
- Impurities (identity and concentrations): Not reported
- Composition of test material, percentage of components: Not reported
- Isomers composition: Not reported
- Purity test date: Not reported
- Lot/batch No.: Exp. I-070518
- Expiration date of the lot/batch: Not reported
- Radiochemical purity (if radiolabelling): Not applicable
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable
- Stability under test conditions: The test samples were tested for stability without prior mixing (sonication) of the test sample bottles to assess for losses due to adsorption and/or insolubility.
The bottom level light and dark and middle level dark samples were just above the normal acceptance limit of 120%. However as most of the other values were just below 120% the difference is not considered significant and the test samples have been considered to be stable in the test medium.
The 0.10 mg/l unsonicated stability vessel showed evidence of insolubility or adherence to glass.
- Storage condition of test material: room temperature in the dark
- Other: Not reported.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not reported.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 30 July 2007 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the funnel three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.8 g/l prior to use.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

7.1 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
Solution a KH2PO4 8.50 g/l
K2HPO4 21.75 g/l
Na2HPO4.2H2O 33.40 g/l
NH4Cl 0.50 g/l

pH = 7.4

Solution b CaCl2 27.50 g/l
Solution c MgSO4.7H2O 22.50 g/l
Solution d FeCl3.6H2O 0.25 g/l

To 1 litre (final volume) of purified water* was added the following volumes of solutions a – d.

10 ml of Solution a
1 ml of Solution b
1 ml of Solution c
1 ml of Solution d

recommended in the OECD Guidelines.

- Additional substrate:
None

- Solubilising agent (type and concentration if used):
- Test temperature:
The test was carried out in a temperature controlled room at approximately 21deg C, in darkness.

- pH:
The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH 320 pH meter.
Please see Table pH Values of the Test Preparations on Day 28

- pH adjusted:
no

- CEC (meq/100 g):

- Aeration of dilution water:
Not recorded

- Suspended solids concentration:
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/l.

- Continuous darkness:
yes

- Other:
In an initial experiment conducted at a concentration of 10 mg C/l inorganic carbon (IC) values in the test material vessels were significantly lower than those in the control vessels thereby suggesting that the test material was exhibiting an inhibitory effect on the activated sewage sludge micro-organisms.

Therefore, following the recommendations of the Test Guidelines, in the definitive test the test material at a reduced concentration of 5 mg C/l was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21deg C for 28 days.
For the purpose of the test, the test material was dissolved directly in culture medium. An amount of test material (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 30 minutes and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (213 ml) of this stock solution was dispersed in inoculated culture medium and the volume adjusted to 3 litres to give a final concentration of 7.1 mg/l, equivalent to 5 mg carbon/l. The volumetric flask containing the test material was inverted several times to ensure homogeneity of the solution.
Data from the control vessels was shared with similar concurrent studies.

Analysis of the concentration, homogeneity and stability of the test material in the test preparations were not appropriate to the Test Guideline.

TEST SYSTEM

The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
c) The test material, in duplicate, in inoculated culture medium to give a final concentration of 5 mg carbon/l.
d) The test material plus the standard material in inoculated culture medium to give a final concentration of 15 mg carbon/l to act as a toxicity control (one vessel only).

Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/l. The test was carried out in a temperature controlled room at approximately 21C, in darkness.

Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 ml of culture medium and 23.7 ml of inoculum and aerated overnight. On Day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.

The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute and stirred continuously by magnetic stirrer.

The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.

The CO2 produced by degradation was collected in two 500 ml Dreschel bottles containing 350 ml of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.


SAMPLING
- Sampling frequency:
CO2 analysis
Samples (2 ml) were taken from the first CO2 absorber vessel on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29.
The samples taken on Days 0, 1, 2, 3, 6, 8, 10, 14, 16, 20, 22, 24, 27, 28 and 29 were analysed for CO2 immediately.

DOC analysis
On Days 0 and 28 samples (20 ml) were removed from all culture vessels and centrifuged (3500 rpm, 15 minutes) prior to DOC analysis.

- Sampling method:
Not recorded

- Sterility check if applicable:
Not applicable

- Sample storage before analysis:
CO2 analysis
The samples taken on Days 12 and 18 were stored at approximately 20deg C. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed that the level of degradation of the test material did not significantly increase during this time and therefore additional analyses were considered to be unnecessary.

DOC analysis
Samples used immediately upon sampling.

- Other:


CONTROL AND BLANK SYSTEM
- Inoculum blank:
A control, in duplicate, consisting of inoculated culture medium.

- Abiotic sterile control:
Not recorded

- Toxicity control:
For the purposes of the test a toxicity control, containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the test.

An aliquot (213 ml) of the test material stock solution was dispersed in inoculated culture medium along with an aliquot (51.4 ml) of the sodium benzoate stock solution. The volume was adjusted to 3 litres to give a final concentration of 7.1 mg test material/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 15 mg carbon/l.

- Other:
Standard Material
For the purposes of the test a standard material, sodium benzoate (C6H5COONa) (Sigma Lot No 035K0151), was used. An initial stock solution of 1000 mg/l was prepared by dissolving the standard material directly in culture medium with the aid of ultrasonication for approximately 2 minutes, and a 51.4 ml aliquot added to the test vessel to give a final test concentration of 17.1 mg/l, equivalent to 10 mg carbon/l. The volumetric flask containing the standard material was inverted several times to ensure homogeneity of the solution.

Data from the standard material vessels was shared with similar concurrent studies.

STATISTICAL METHODS: Not reported.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

The results obtained from the preliminary investigational work showed that whilst the test material did not adsorb to filter matrices or to activated sewage sludge, a slightly higher measured concentration was obtained in the centrifuged sample plus inoculum compared to the filtered sample plus inoculum. Therefore for the purpose of the study the samples taken for DOC analysis were centrifuged to remove the suspended solids present as a precautionary measure.

Test performance:

The total CO2 evolution in the control vessels on Day 28 was 23.53 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The IC content of the test material suspension in the mineral medium at the start of the test (see Table 3) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.

The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

44

Sampling time:

28 d

Remarks on result:

other: No ready biodegradable

Details on results:

Inorganic carbon values for the test material, standard material, toxicity control and control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and standard materials and the toxicity control are given in Table 2. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3, and the results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given in Table 4.

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test material.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of standard material Replicate R2. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

The test material attained 44% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Analysis of the test media from the test material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), see Table 4, gave percentage degradation values of 58% and 52% respectively for the test material Replicates R1 and R2 and 95% for the toxicity control. These degradation rates calculated from the results of the DOC analyses were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of test material/sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occurring. Sodium benzoate attained 100% and 98% degradation respectively for Replicates R1 and R2 calculated from the results of the DOC analyses. These degradation rates were similar to those calculated from inorganic carbon analysis.
Observations made throughout the test period (see Table 6) showed the contents of the control vessels to be light brown dispersions and the contents of the standard material vessels were light brown dispersions with no undissolved standard material visible. The contents of the test material vessels were light brown dispersions with no undissolved test material visible. The toxicity control vessel was observed to contain a light brown dispersion with no undissolved test or standard material visible.

BOD5 / COD results

Results with reference substance:

The toxicity control attained 31% degradation after 14 days and 71% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test.
Sodium benzoate attained 97% degradation after 14 days and 113% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Degradation values in excess of 100% were considered to be due to an increase in the numbers of viable micro-organisms in the standard material vessels as a result of the readily biodegradable nature of sodium benzoate. This effect occurs due to the micro-organisms utilizing the standard material as a carbon source for cellular growth resulting in a greater number of viable micro-organisms in the standard material vessels when compared to the control vessels. This increased number of micro-organisms in the standard material vessels gave rise to increased respiration rates and hence background CO2 evolution was greater than in the control vessels. This increase in background CO2 evolution resulted in biodegradation rates in excess of 100%.

Any other information on results incl. tables

Table 1               Inorganic Carbon Values on Each Analysis Occasion

Day	Control (mg IC)				Sodium Benzoate (mg IC)				Test Material (mg IC)				Test Material plus Sodium Benzoate Toxicity Control (mg IC)
	R1		R2		R1		R2		R1		R2		R1
	Abs1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	2.10	1.98	2.10	1.87	2.80	1.87	2.10	1.98	1.87	2.45	1.75	2.45	2.22	2.45
1	5.57	-	4.87	-	8.12	-	8.00	-	4.87	-	5.92	-	5.80	-
2	7.61	-	6.57	-	22.84	-	22.60	-	7.61	-	7.96	-	20.30	-
3	6.99	-	5.85	-	23.28	-	33.37	-	7.22	-	7.57	-	19.49	-
6	11.63	-	9.23	-	29.98	-	37.39	-	12.42	-	10.83	-	24.05	-
8	12.58	-	10.09	-	31.51	-	38.31	-	11.22	-	10.54	-	23.80	-
10	13.52	-	10.37	-	33.35	-	47.43	-	12.51	-	10.70	-	23.77	-
14	15.59	-	10.58	-	34.85	-	49.77	-	15.14	-	13.58	-	26.83	-
16	15.94	-	13.28	-	45.04	-	50.13	-	17.15	-	16.71	-	30.88	-
20	16.07	-	15.20	-	46.80	-	51.28	-	22.74	-	16.40	-	32.91	-
22	17.39	-	15.32	-	47.70	-	51.29	-	25.65	-	18.58	-	39.45	-
24	17.50	-	17.71	-	51.30	-	50.97	-	25.05	-	22.57	-	40.72	-
27	20.18	-	19.64	-	52.16	-	53.99	-	26.51	-	24.90	-	44.86	-
28	19.31	-	19.20	-	52.59	-	55.57	-	24.85	-	24.96	-	48.21	-
29	20.14	2.44	19.82	2.09	53.53	2.09	54.06	2.09	26.50	2.78	25.65	2.78	50.88	3.48

Table 2               Percentage Biodegradation Values

Day	% Degradation Sodium Benzoate	% Degradation Test Material	% Degradation Test Material plus Sodium Benzoate Toxicity Control
0	0	0	0
1	9	1	1
2	52	5	29
3	73	7	29
6	78	8	30
8	79	0	28
10	95	0	26
14	97	8	31
16	110	15	36
20	111	26	38
22	110	38	51
24	112	41	51
27	111	39	55
28	116	38	64
29*	113	44	71

*Day 29 values corrected to include any carry-over of CO₂detected in Absorber 2

Table 3               Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel	Total Carbon* (mg/l)	Inorganic Carbon* (mg/l)	IC Content (% of TC)
Sodium Benzoate 10 mg C/lR1	10.82	0.04	0
Sodium Benzoate 10 mg C/l R2	9.63	-1.77	0
Test Material 5 mg C/l R1	3.11	-1.72	0
Test Material 5 mg C/l R2	2.94	-1.75	0
Test Material plus Sodium Benzoate Toxicity Control 15 mg C/l	11.64	-1.64	0

*Corrected for control values. Negative values are due to measured concentrations being less than control values

Table 4               Dissolved Organic Carbon (DOC) Values in the Culture Vessels on Days 0 and 28

Test Vessel	DOC*Concentration
	Day 0		Day 28
	mg C/l	% of Nominal Carbon Content	mg C/l	% of Initial Carbon Concentration	% Degradation
Sodium Benzoate 10 mg C/l R1	10.78	108	0.00	0	100
Sodium Benzoate 10 mg C/l R2	11.40	114	0.23	2	98
Test Material 5 mg C/l R1	4.83	97	2.04	42	58
Test Material 5 mg C/l R2	4.69	94	2.23	48	52
Test Material plus Sodium Benzoate Toxicity Control 15 mg C/l	13.27	88	0.68	5	95

*Corrected for control values.

Table 5               Observations on the Test Preparations Throughout the Test Period 

Test Vessel		Observations on Test Preparations
		Day 0	Day 6	Day 13	Day 20	Day 27
Control	R1	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
	R2	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
Standard Material	R1	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible
	R2	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible
Test Material	R1	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible
	R2	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible	Light brown dispersion, no undissolved test material visible
Toxicity Control		Light brown dispersion, no undissolved test or standard material visible	Light brown dispersion, no undissolved test or standard material visible	Light brown dispersion, no undissolved test or standard material visible	Light brown dispersion, no undissolved test or standard material visible	Light brown dispersion, no undissolved test or standard material visible

R₁– R₂= Replicates 1 and 2

Abs= CO₂absorber vessel

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test material attained 44% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Executive summary:

Introduction.

A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO₂Evolution Test" referenced as Method C.4-C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 Paragraph (m).

Methods.

In an initial experiment conducted at a concentration of 10 mg C/l inorganic carbon (IC) values in the test material vessels were significantly lower than those in the control vessels thereby suggesting that the test material was exhibiting an inhibitory effect on the activated sewage sludge micro-organisms.

Therefore, following the recommendations of the Test Guidelines, in the definitive test, the test material at a reduced concentration of 5 mg C/l was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

Results.

The test material attained 44% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.